For the treatment of β-globin gene defects, a homologous recombination-mediated gene correction approach would provide advantages over random integration-based gene therapy strategies. However, "neighborhood effects" from retained selectable marker genes in the targeted locus are among the key issues that must be taken into consideration for any attempt to use this strategy for gene correction. An Ala-to-lle mutation was created in the β6 position of the mouse β-major globin gene (β61) as a step toward the development of a murine model system that could serve as a platform for therapeutic gene correction studies. The marked β-major gene can be tracked at the level of DNA, RNA, and protein, allowing investigation of the impact of a retained phosphoglycerate kinase (PGK)-neo cassette located between the mutant β-major and β-minor globin genes on expression of these 2 neighboring genes. Although the PGK-neo cassette was expressed at high levels in adult erythroid cells, the abundance of the β61 mRNA was indistinguishable from that of the wildtype counterpart in bone marrow cells. Similarly, the output from the β-minor globin gene was also normal. Therefore, in this specific location, the retained, transcriptionally active PGK-neo cassette does not disrupt the regulated expression of the adult β-globin genes.